Bisphenol A alters early oogenesis and follicle formation in the fetal ovary of the rhesus monkey Patricia A. Hunta,1, Crystal Lawsona, Mary Gieskea, Brenda Murdocha, Helen Smitha, Alyssa Marrea, Terry Hassolda, and Catherine A. VandeVoortb aSchool of Molecular Biosciences and Center for Reproductive Biology, Washington State University, Pullman, WA 99164; and bDepartment of Obstetrics and Gynecology and California National Primate Research Center, University of California, Davis, CA 95616 Edited* by Joan V. Ruderman, Harvard Medical School, Boston, MA, and approved August 24, 2012 (received for review May 9, 2012) Widespread use of the endocrine disrupting chemical bisphenol A a model for developmental toxicology it has distinct advantages (BPA) in consumer products has resulted in nearly continuous human (12) because pregnancy in primates and rodents differs in several exposure. In rodents, low-dose exposures have been reported to important respects, including placentation (13), placental pro- adversely affect two distinct stages of oogenesis in the developing tein products (14), and fetal adrenal function (15). Of particular ovary: the events of prophase at the onset of meiosis in the fetal relevance for studies of endocrine disrupting chemicals, the ovary and the formation of follicles in the perinatal ovary. Because levels of estrogen maintained throughout pregnancy in rhesus – these effects could influence the reproductive longevity and success females are similar to those in humans (16 18), which is not the of the exposed individual, we conducted studies in the rhesus case in mice. monkey to determine whether BPA induces similar disturbances in Previous studies in mice suggest that low-level BPA exposure disrupts oogenesis at multiple stages. It disturbs the behavior of the developing primate ovary. The routes and levels of human chromosomes at the onset of meiosis in the fetal ovary, disrupts exposure are unclear; hence, two different exposure protocols were the packaging of meiotically arrested oocytes into follicles in the used: single daily oral doses and continuous exposure via subdermal newborn ovary, and affects the final stages of oocyte maturation in implant. Our analyses of second trimester fetuses exposed at the the adult ovary [(19–21), reviewed in ref. 5)]. In the present study, time of meiotic onset suggest that, as in mice, BPA induces subtle we investigated whether defects in the fetal stages of oogenesis disturbances in the prophase events that set the stage for chromo- might also be apparent in BPA-exposed rhesus females. GENETICS some segregation at the first meiotic division. Our analyses of third- The studies presented here were conducted in conjunction with trimester fetuses exposed to single daily oral doses during the time pharmacokinetic studies of female rhesus monkeys (9). Initial − − of follicle formation revealed an increase in multioocyte follicles studies using single oral doses of 400 μg·kg 1·d 1 administered to analogous to that reported in rodents. However, two unique phe- nonpregnant females demonstrated rapid conjugation (in- notypes were evident in continuously exposed animals: persistent activation) of BPA, with peak serum levels of 2–5 ng/mL attained unenclosed oocytes in the medullary region and small, nongrowing 1–2 h after ingestion and a rapid decline thereafter. Because the oocytes in secondary and antral follicles. Because effects on both dose was high (∼8 times the current FDA “safe” dose) but peak stages of oogenesis were elicited using doses that yield circulating levels closely approximated levels observed in human studies levels of BPA analogous to those reported in humans, these findings (reviewed in refs. 22 and 23), we concluded that human exposure raise concerns for human reproductive health. must be significantly higher than assumed, not primarily restricted to oral routes, and nearly constant (9). Thus, in addition to using an oral dosing strategy, we developed a protocol using the im- isphenol A (BPA) is a synthetic chemical that has endocrine plantation of controlled-release Silastic capsules to achieve sus- disrupting properties. Owing to its high-volume production B tained low-level exposure. and widespread use in consumer products, including canned foods, The timing of exposure using both protocols was designed to pressure-printed receipts, dental sealants, and plastic products, mimic the developmental windows that elicited effects in the ovary humans are exposed to BPA on a daily basis. During the past 15 y, in mice: an early exposure during the second trimester of pregnancy adverse effects of low-dose exposures have been reported in [gestational day (GD) 50–100], when germ cell differentiation and hundreds of studies of experimental animals (reviewed in refs. 1 meiotic entry occur, and a late exposure during the third trimester and 2), and human studies reporting adverse effects are steadily (GD 100–term), when follicle formation takes place. We report increasing (reviewed in ref. 3). here the results of meiotic studies of BPA-exposed and control Environmental exposures that affect the developing re- animals from the early exposure cohort and studies of follicle productive tract or influence gamete production may compromise morphology and the first wave of follicle growth from the late ex- fertility and, consequently, findings from studies of rodents ex- posure cohort. Our results confirm previous findings in rodents, posed to BPA are of great concern. Fetal and neonatal BPA exposures reportedly affect the developing reproductive tract of indicating that the early stages of oocyte development in the rhesus both males and females, several distinct stages of oogenesis in the monkey are vulnerable to disturbance by BPA and suggesting that developing ovary, testosterone levels and sperm counts in the adult fetal exposures may adversely affect the reproductive potential of male, and the fertility of females exposed in utero (reviewed in adult female primates, including humans. – refs. 4 7). Results Despite growing evidence of harm, the relevance of findings from rodent studies has been challenged on the grounds that dif- The results presented here represent one of several studies of the ferences in BPA metabolism may result in different responses in same cohort of females, among them pharmacokinetic studies rodents and humans to the same doses of BPA (reviewed in ref. 8). Recent pharmacokinetic studies, however, have provided direct evidence that, despite differences in metabolism, the pharmaco- Author contributions: P.A.H. and C.A.V. designed research; P.A.H., C.L., M.G., B.M., H.S., kinetics are extraordinarily similar in rodents, nonhuman primates, A.M., and C.A.V. performed research; P.A.H., C.L., T.H., and C.A.V. analyzed data; and and humans (9). Nevertheless, given the seriousness of the possible P.A.H., T.H., and C.A.V. wrote the paper. effects of BPA on the reproductive potential of the female, studies The authors declare no conflict of interest. in an animal model with greater similarity to the human are *This Direct Submission article had a prearranged editor. clearly warranted. 1To whom correspondence should be addressed. E-mail: [email protected]. The rhesus monkey has been recognized as a superior model This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. for human reproductive physiology for many years (10, 11). As 1073/pnas.1207854109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1207854109 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 (9) and studies of other organ systems, including the mammary oral dose. Although MLH1 counts were higher in both exposed gland (24). The early and late exposure protocols were designed animals than in the single control, the limited data precluded to mimic the developmental windows reported to elicit effects on meaningful analysis. A sufficient number of pachytene cells for oogenesis in mice (reviewed in ref. 5). For both exposures, we analysis were available, however, from animals exposed continu- examined effects resulting from single daily oral doses of BPA ously to BPA via Silastic implants. Consistent with previous findings consumed by the mother and of continuous low-level exposure in mice (19), a highly significant increase in mean MLH1 values per delivered via maternal subdermal implant. cell was observed in oocytes from exposed compared with placebo- For animals receiving oral doses, mean levels of bioactive (un- treated fetuses (50.4 ± 7.0 for 33 cells from exposed females and conjugated) BPA in maternal serum close to the time of ovarian 42.2 ± 5.9 for 70 cells from controls; t = 6.8; P < 0.001; Fig. 1). tissue collection were 0.51 ± 0.20 ng/mL for second-trimester In the mouse, BPA induced synaptic defects (i.e., defects in- pregnancies and 0.31 ± 0.13 ng/mL for third-trimester pregnan- volving one or several chromosome pairs in an otherwise normal cies. These values were obtained 4 h after administration of the last pachytene cell) and end-to-end associations that almost exclusively maternal oral dose; in nonpregnant females, this time point was involved centromeric regions of nonhomologous chromosomes closest to the average level during the 24 h following a single oral (19). To determine whether BPA exposure in the rhesus monkey dose (9). For animals in the implant exposure group, mean levels induced similar defects, cells judged to be at pachytene on the basis of bioactive BPA in maternal blood at the time of ovary collection of SYCP3 staining were scored. As in the human female (27), the were 0.45 ± 0.23 ng/mL for second-trimester fetuses and 0.90 ± incidence of synaptic defects in control females was high compared 0.13 ng/mL for third-trimester fetuses; these values represent the with that in the mouse, with over one-third of cells displaying some sustained levels achieved using the implant paradigm and are in type of synaptic defect (Fig. 2). Against this high background, close agreement with levels reported in human maternal serum a slight decrease in the proportion of normal pachytene cells was (reviewed in ref.